Various factors for determining skin or hair color have been reported. Among others, the amount or quality of melanin existing in the epidermis is considered to greatly contribute to skin or hair color. Specifically, it is said that melanin produced in an organelle known as melanosome in a pigment cell (melanocyte) is transferred to keratinocytes in the epidermis or follicle and is then spread over the epidermis and hair as a whole, and that formation of skin or hair color is significantly influenced by such movement of melanin. Such melanin generated by melanocytes has been long known as a factor associated with the skin or hair color of individuals.
Melanocyte, which is a cell associated with the biosynthesis of melanin, contains a melanosome which is a unique lysosome-related organelle derived from endosome. In this melanosome, melanin is synthesized via a catalytic pathway having tyrosine as a precursor. Such a melanosome receives various gene products mainly sorted from trans-Golgi network, and as a result, it has properties shared by lysosome (e.g. internal low pH condition). Nevertheless, structural proteins including a series of melanin synthetases and the related tyrosinase, dopachrome tautomerase and Pmel-17 (GP100) are transferred only to the melanosome, and as a result, the melanosome has a specific property of synthesizing melanin. In the process of generation of skin color, a pigmented melanosome becomes matured as a result of control by various membrane transport factors such as Rab family members, and subsequently, the matured melanosome is transferred to a dendrite and is then transferred to keratinocytes adjacent thereto.
Conventional skin lightening agents have been developed, while mainly targeting generation of melanin in melanocytes. In Non-Patent Document 1, for example, ascorbic acid, arbutin, Kojic acid and the like have been reported as skin lightening agents having an action to inhibit the enzymatic activity of tyrosinase, an enzyme involved in the conversion of tyrosine, which is a melanin precursor, to melanin, so as to suppress generation of melanin.
On the other hand, it has also been reported that a difference is found in the mode or the maturation state of melanin in keratinocytes, according to a difference in skin color (Non-Patent Document 2). That is to say, it is considered that the dynamics of melanin in keratinocytes would play a certain role for determining skin color. According to previous reports, it has been suggested that a receptor molecule PAR-2 be associated with uptake of melanin into keratinocytes (phagocytosis), and that skin color be controlled by regulating such activity (Non-Patent Documents 3 to 8). Moreover, it has also been suggested that protein molecules Dynein or Dynactin be associated with localization of melanin, which has been transferred into keratinocytes, in the keratinocytes (Non-Patent Documents 9 and 10). Furthermore, it has also been suggested that MyoX, a protein molecule involved in formation of a cell structure known as Filopodia, be associated with the transport of melanin from melanocytes to keratinocytes, and from keratinocytes to keratinocytes (Non-Patent Document 11). However, mechanisms regarding the uptake of melanin generated in melanocytes (melanosomes) by keratinocytes, transport, and metabolism have not yet been sufficiently elucidated, and the role of melanin for skin color has not been verified, either.
Recently, as a result of studies using keratinocytes derived from different ethnic groups, it has been suggested that, in terms of the metabolic capacity of melanin taken up, there be a difference among ethnic groups (Non-Patent Document 12). Non-Patent Document 12 reports that melanin is easily degraded in epidermal cells derived from a Caucasian subject, using an evaluation system in which a fluorescent substance-labeled melanin is taken up into epidermal cells and the disappearance of fluorescence in the epidermal cells is then analyzed. However, this document does not refer to any mechanisms contributing to degradation or specific factors.
Autophagy generally means a process in which an intracellular substance such as an organelle is transferred into a lysosome and is then degraded (Non-Patent Documents 13 to 15). It has been known that autophagy includes several types. Among such types, a main pathway, which has been studied most intensively, is macroautophagy. In general, the term “autophagy” indicates such macroautophagy. Autophagy is induced by: various physiological stress such as fasting, hypoxia, energy depletion, endoplasmic reticulum stress, or high temperature; hormonal stimulation; drugs (rapamycin, fluspirilene, trifluoperazine, pimozide, nicardipine, niguldipine, loperamide, amiodarone, verapamil, minoxidil, clonidine, etc.); immune signaling; infection with bacteria, virus and parasite; and disease such as acute pancreatitis or heart disease. Since autophagy-related phenomena have been observed in a wide range of species including yeasts and mammals, autophagy is considered to play an extremely important role for organisms. In fact, it has been reported so far that autophagy is involved in cancer, neurodegenerative disease, inflammation, immunization, aging, and the like (Non-Patent Document 16).
Autophagy consists of a series of processes caused via a special organelle known as an autophagosome. In the autophagic pathway, an isolation membrane or a vesicle called phagophore is first generated in a cytoplasm. The phagophore gradually elongates and then encloses a portion of the cytoplasm, so as to form an autophagosome, which is a sac-like structure having a double membrane. Subsequently, the outer membrane of the autophagosome is fused with a lysosome to form an autolysosome, and in the autolysosome, the enclosed substance is degraded together with the inner membrane of the autophagosome. The degradation product generated as a result of autophagy is reused in a cell. The autophagic pathway is mediated by a plurality of steps such as the generation and elongation of phagophores, formation of autophagosomes, the fusion of autophagosomes with lysosomes, and formation of autolysosomes and degradation of the internal substance, and further, many factors are involved in each step. Main factors are proteins involved in the formation of phagophores and autophagosomes, which are collectively referred to as ATG (Autophagy-related proteins). To date, 30 or more ATGs have been reported (Non-Patent Document 11).
However, it has not been known so far that autophagy is associated with the dynamics of melanin in keratinocytes, such as the uptake, accumulation and degradation of melanin.